Oropharyngeal cancer (OPC) incidence is significantly increasing in men and is four- to fivefold higher in men compared to women.1 The majority of OPCs are diagnosed at late stages, requiring intensive treatment that leads to significant long-term morbidity. HPV infection has replaced smoking as the primary cause of OPC, with ~70% of OPCs caused by HPV infection, predominantly with HPV type 16. Unlike other cancers, there is no reliably detected pre-neoplastic lesion to utilize in early detection interventions, and oral Pap smears are ineffective in detecting early OPC lesions. Therefore, reliance on a biomarker approach is necessary for the development of OPC screening interventions. The molecular pathway to OPC is similar to that of cervical cancer: a subset of HPV infections persist (obligate precursor to lesion development), followed by HPV integration into the host genome, overexpression of HPV oncoproteins E6 and E7 leading to p16 overexpression, and nonrandom chromosomal gains. As HPV-related OPC and cervical cancer are biologically similar, our most effective approach in developing OPC biomarkers is to leverage knowledge informed by cervical cancer biomarker studies. Currently, the accepted standard specimen for detecting oral HPV is the oral gargle. Here, we propose an innovative approach for the development of an OPC early detection intervention by applying cervical cancer biomarkers to a new specimen and cancer: the oral gargle and OPC. Rapid translation to the clinic is possible, as we propose a specimen type that is non-invasive, with sufficient material to support multiple biomarker measurements, and a panel of markers with proven utility at the cervix, for which our preliminary data show feasibility. Our goal is to develop a sensitive and specific biomarker panel that is diagnostic of OPC. The hypothesis is that oral gargle biomarkers related to cellular disruption, HPV viral characteristics, and host cell epigenetic alterations can be used as surrogates for the presence of these markers in tumor tissue and can distinguish OPC cases from cancer-free men. In this developmental R21 application, we propose to enroll 100 OPC cases (pre-treatment) and a frequency age-matched set of cancer-free men (at risk population) to address the following Specific Aims: 1) Among OPC cases, assess the correlation of biomarkers (HPV genotype, p16, HPV 16 methylation of L1, E2, E5 proteins, and host methylation) measured in tumor and oral gargle specimens; 2) Evaluate the most promising oral gargle biomarkers with high correlation in Aim 1 in cancer-free men to establish cut-points (thresholds for positivity) for the detection of OPC, and assess the prevalence of individual biomarkers among cases and cancer-free men; and 3) Determine the optimal combination of oral gargle biomarkers to detect OPC. In secondary aims, we will identify chromosomal aberrations, host gene methylation signatures, and innate immune factors that may serve as supplemental diagnostic OPC markers.